Developments in water processing technology have significantly decreased the incidence of illness from waterborne pathogenic organisms. However, isolated cases of illnesses owing to such organisms are common, and occasionally large-scale outbreaks of illness occur. As an example, severe outbreaks of gastroenteritis have been caused by the environmentally resistant intestinal protozoan parasites Cryptosporidium, Giardia, Microsporidia, and Cyclospora. These organisms are often waterborne, existing in very dilute densities in public water supplies, and many have only recently been recognized as causing a health problem. As an example, Cryptosporidium has been linked to waterborne outbreaks of gastroenteritis only since 1981, when widespread cases of acute gastroenteritis in a Texas community were traced to the presence of Cryptosporidium in the drinking water supply. Numerous other cities have experienced outbreaks since then. Cryptosporidium oocysts are exemplary of waterborne protozoan parasites in that they can survive for long times in water and are resistant to routine water treatment methods such as chlorination. The presence in source waters of even small numbers of Cryptosporidium oocysts is a matter of concern because the infective dose for humans is possibly as low as one oocyst. Additionally, exposure to protozoan parasites or other waterborne pathogens can be life-threatening, particularly to infants, the elderly, and people whose immune systems are impaired. In fact, many waterborne pathogens are only now receiving significant scientific attention after having been traced to fatalities in this population, e.g., Microsporidia.
Clearly, the detection of pathogenic organisms such as Cryptosporidium in water supplies is a critical societal problem. The central issue in detection of such organisms is how to concentrate dilute densities of the organisms from large volumes of water so as to detect their presence.
Regarding protozoan parasites such as Cryptosporidium, the method currently recommended by the Environmental Protection Agency (EPA) and the American Society for Testing and Materials (ASTM) concentrates the parasites by sampling 100 liters (or more) of water through a polypropylene yarn cartridge filter. As summarized in Nieminski et al., Applied and Environmental Microbiology, 61(5): 1714-1719, 1995, in the ASTM method, after sampling, particulates from the cartridge filter are extracted by cutting the filter apart and washing the fibers. The extracted particulates are then concentrated by centrifugation. The concentrated particulates are then processed to selectively concentrate parasite cysts and oocysts by floatation in 50-mL tubes on a Percoll-sucrose gradient. Particulates recovered at the interface of the Percoll-sucrose gradient are stained with fluorescently tagged antibodies on 25-mm-diameter, 0.2 .mu.m-pore-size cellulose acetate filters. After mounting on slides, the membrane filters are scanned with an epifluorescent microscope for objects having the size, shape and fluorescence characteristic of Cryptosporidium oocysts. On finding such objects, the microscope optics are switched to phase contrast to look for internal morphological characteristics inside the detected organisms. Organisms determined to meet the fluorescence detection criteria are counted as presumptive Cryptosporidium oocysts. Organisms with the right fluorescence characteristics and shown to have the respective internal morphological characteristics are counted as confirmed Cryptosporidium oocysts.
However, the ASTM method is costly and consumes substantial time and labor. It is also not very effective since losses of oocysts occur throughout the procedure. Large numbers of oocysts pass through the filter, or adhere to the filter material and are not recovered. Losses also occur during centrifugation because oocysts are destroyed or resuspended during removal of the supernatant fluid. During a recent blind test of commercial laboratories in the United States, spiked samples were submitted to sixteen laboratories to evaluate their ability to recover and detect Cryptosporidium oocysts using the ASTM method. Six laboratories--over one third of the total--failed to recover any parasites, and the remaining ten had an average recovery rate of only 2.8%. Aldom et al., Letters in Applied Microbiology 20: 186-187, 1995. A more detailed description of losses of Cryptosporidium oocysts during the detection procedure can be found in LeChevallier et al., Applied and Environmental Microbiology 61 (2): 690-697, 1995.
Several studies have been conducted on techniques to improve the efficiencies of methods for concentrating the dilute protozoa. The general protocol in such studies is to spike a water sample with a known amount of Cryptosporidium oocysts and determine the percentage of oocysts recovered for each method tested.
A number of studies evaluated other types of filtration, including vortex-flow, cross-flow, and sand column filtration. Whitmore et al., Wat. Sci. Tech. 27 (3-4): 69-76, 1993. In the Whitmore et al. study, the vortex-flow filtration technique gave fairly consistent recoveries of 30% to 40%. While this retention rate is superior to that of the ASTM method, the comparatively long process times would prevent the use of this method for monitoring purposes. The crossflow filtration module also gave relatively good recoveries (approximately 40-80%) at moderately high flow rates. The laboratory scale sand columns evaluated gave satisfactory retention within the column material at low flow rates, but were judged inadequate for monitoring purposes because of the poor retention of oocysts within the column matrix at realistic flow rates. Another study tested a filter matrix dissolution method to recover Cryptosporidium oocysts from water. The average recovery rate observed was 70.5%. Aldom et al., Letters in Applied Microbiology 20: 186-187, 1995. Cryptosporidium oocysts have also been concentrated from water by "sweeping" water with a settling calcium carbonate precipitate. Vesey et al., Journal of Applied Bacteriology 75: 82-86, 1993. The Vesey et al. study resulted in a 68% recovery of oocysts from seeded samples of deionized, tap, and river water.
Continuous flow bowl centrifugation has also been studied as a means to concentrate dilute concentrations of Cryptosporidium oocysts. An exemplary continuous flow bowl centrifuge commonly referred to as a Latham bowl centrifuge is shown at 20 in FIG. 1. Liquid is introduced into the end (top or bottom) of a spinning bowl 22 from an axially-directed input line 24 with length r1 located near the rotational axis of the bowl 22. As the liquid flows up the height h of the bowl 22, centrifugal force forms a density gradient in the liquid with the densest (heaviest) matter (sand or other detritus) located near the wall of the bowl 22 and the lighter matter (e.g., water) being located closer to the rotational axis. As liquid is introduced into the bowl 22, the lighter portion of the liquid is emptied at the same flow rate from an axially-situated and axially-directed exit line 26 with length r2 which exits the end of the bowl 22 opposite the input line 24. Continuous flow bowl centrifugation has been used in conjunction with heating to kill bacteria present in beverages, as described by U.S. Pat. No. 3,217,982 to Wilsmann et al. However, studies evaluating use of a continuous flow bowl centrifuge for concentrating protozoan parasites have not described increases in recovery rates. The Whitmore et al. studies cited above evaluated use of a bowl-type continuous flow centrifuge for concentration of oocysts and reported recovery between 11% to 31.2%. Goatcher et al., American Society of Macrobiology Abstracts Q-212, 1995 also tested a bowl-type continuous flow centrifuge system and reported obtaining recovery rates of between 2 to 20 times those observed with conventional filtration methods; therefore, such recovery rates are still well under 50%.
In view of the importance of protecting water supplies from pathogenic organisms such as protozoan parasites, and the lack of apparata or methods for concentrating and/or detecting such organisms with high recovery rates, it is clearly desirable to develop improved methods and apparata for reliably concentrating waterborne pathogenic organisms.